MDR3 P-glycoprotein, a Phosphatidylcholine Translocase, Transports Several Cytotoxic Drugs and Directly Interacts with Drugs as Judged by Interference with Nucleotide Trapping

Abstract

The human MDR3 gene is a member of the multidrug resistance (MDR) gene family. The MDR3 P-glycoprotein is a transmembrane protein that translocates phosphatidylcholine. The MDR1 P-glycoprotein related transports cytotoxic drugs. Its overexpression can make cells resistant to a variety of drugs. Attempts to show that MDR3 P-glycoprotein can cause MDR have been unsuccessful thus far. Here, we report an increased directional transport of several MDR1 P-glycoprotein substrates, such as digoxin, paclitaxel, and vinblastine, through polarized monolayers of MDR3-transfected cells. Transport of other good MDR1 P-glycoprotein substrates, including cyclosporin A and dexamethasone, was not detectably increased. MDR3 P-glycoprotein-dependent transport of a short-chain phosphatidylcholine analog and drugs was inhibited by several MDR reversal agents and other drugs, indicating an interaction between these compounds and MDR3 P-gp. Insect cell membranes from Sf9 cells overexpressing MDR3 showed specific MgATP binding and a vanadate-dependent, Nethylmaleimide-sensitive nucleotide trapping activity, visualized by covalent binding with [␣-32 P]8-azido-ATP. Nucleotide trapping was (nearly) abolished by paclitaxel, vinblastine, and the MDR reversal agents verapamil, cyclosporin A, and PSC 833. We conclude that MDR3 P-glycoprotein can bind and transport a subset of MDR1 P-glycoprotein substrates. The rate of MDR3 Pglycoprotein-mediated transport is low for most drugs, explaining why this protein is not detectably involved in multidrug resistance. It remains possible, however, that drug binding to MDR3 P-glycoprotein could adversely affect phospholipid or toxin secretion under conditions of stress (e.g. in pregnant heterozygotes with one MDR3 null allele). P-glycoproteins (P-gps)1 are 170-kDa glycosylated membrane proteins that actively transport their substrates out of the cell. Two P-gp genes have been identified in humans. The human MDR1 gene encodes a drug transporting P-gp that can actively extrude a range of cytotoxic anticancer drugs from the cytoplasm. Overexpression of this P-gp gene results in a decreased intracellular accumulation of these drugs and renders the cell multidrug resistant (1-3). The second human P-gp gene is MDR3 (also known as MDR2) (4, 5). Attempts to obtain resistance against cytotoxic drugs by transfecting drug-sensitive cell lines with the MDR3 cDNA or its mouse homolog, Mdr2, yielded only negative results (4, 6 -9). Amplification or activation of the MDR3 gene, independent of the closely linked MDR1, has never been found in multidrug-resistant cell lines (10 -12). Nevertheless, two studies suggest a role for MDR3 P-gp in drug transport: 1) analysis of B-cell leukemias showed a correlation between MDR3 overexpression and daunorubicin transport (13, 14), and 2) Kino et al. (15) found low level resistance against the antifungal agent aureobasidin A in yeast transformed with the human MDR3 cDNA.The function of the murine homolog of human MDR3 P-gp, Mdr2 P-gp, became clear when Smit et al. (16,17) generated...

In vivo imaging of hepatic excretory function in the rat by fluorescence microscopy

“…Biliary excretion of ICG was reduced by 90% in Mdr2 À/À (multidrug-resistance polypeptide-2, Mdr2) mice, suggesting that this canalicular transport protein is essential for dye elimination [22]. Rodent Mdr2 and its human homolog MDR3 function as ATP-consuming transport proteins that translocate phospholipids and several xenobiotics from the cytosolic to the luminal leaflet of the canalicular membrane [23]. DY635 has not been applied for in vivo microscopy so far.…”

Bile salt–dependent efflux of cellular phospholipids mediated by ATP binding cassette protein B4

“…32 The resistance of yeast cells to aureobasidin A conferred by ABCB4 can be overcome by vinblastine, verapamil, and cyclosporin A. Transepithelial transport of C6-NBD-PC and digoxin by ABCB4 through LLC-PK1 cells are inhibited by vinblastine, verapamil, and cyclosporin A, indicating an interaction between these compounds and ABCB4. 33 The translocation of NBD-PC in yeast secretory vesicles is mediated by both ABCB1 and ABCB4 and abrogated by verapamil. 14 Our results showed that verapamil almost completely abolished the NaTC-dependent efflux of phospholipids and cholesterol mediated by ABCB4 (Fig.…”

“…Verapamil is a transport substrate for ABCB1, and in addition is a potent inhibitor of ABCB1 and ABCB4. 14,17,[32][33][34] The effect of verapamil on the ABCB4-mediated lipid efflux was examined (Fig. 7).…”

Identification of genes that confer tumor cell resistance to the Aurora B kinase inhibitor, AZD1152

“…Significantly less drug was measured in the cytosol of the resistant line compared to parental SW620 cells by LC-MS analysis ( Figure 3b). PSC-833, a small-molecule inhibitor of MDR1, 17,18 and MDR3, 19 was effective in reversing the resistance of SW620 MDR1/3 cells to AZD1152 HQPA ( Figure 3c). Partial knockdown of MDR1 (B75%) with siRNA partially restored inhibition of histone H3 phosphorylation by AZD1152 HQPA in SW620 MDR1/3 ( Figure 3d), suggesting that upregulation of MDR1 is required for full resistance to AZD1152 HQPA in this model.…”